Abstracts for poster session on Thursday May 19th
P1: Influence of the membrane geometry on the organisation of actomyosin networks reconstituted in GUVs
Marcos Arribas Perez, Gijsje Koenerink
Delft University of Technology, The Netherlands
Animal cytokinesis is a dynamic and tightly regulated process by which a contractile ring-like structure of actomyosin filaments pulls from the plasma membrane and constricts the cell at its mid-plane. However, the precise composition of the actomyosin ring and the mechanisms that promote its formation and force generation are still poorly understood. In this context, bottom-up reconstitution of minimal division machineries represent simpler experimental systems which contribute to gain new insights into biochemical and biophysical aspects of cell division and serve as a potential system to achieve controlled division of synthetic cells. So far, most efforts have focused on understanding how the actin cytoskeleton induces changes in the membrane shape. Nevertheless, previous studies have shown that the formation of actomyosin rings in confinement and the initiation of the cleavage furrow ingression are influenced by the size and curvature of the compartment and the cortical tension distribution [1-3].
Here, using eDICE we co-encapsulate actin, actin-binding proteins and myosin in GUVs to reconstitute synthetic contractile actomyosin networks. Then, to experimentally test the role of the geometry of GUVs in the organisation and contractility of the reconstituted actomyosin networks, we employ different strategies to impose membrane deformations (e.g micropipette aspiration and curvature-inducing proteins) in combination with fluorescence microscopy imaging to monitor the dynamic response of the cytoskeleton and the membrane to the new conditions.
References: [1]. Miyazaki, M., et al. Nat. Cell Biol., 17, 480– 489 (2015). [2]. Bashirzadeh, Y. et al. Commun Biol 4, 1136 (2021). [3]. Poirier, et al. PLoS Comput. Biol. 8, e100.
P2: Metalloregulation leads to the different cell behaviors in artificial cells
Taniya Chakraborty, Sayuri Higashi, Seraphine Wegner
Institute of Physiological Chemistry and Pathobiochemistry/ University of Münster, Germany
Inside sophisticate living cells, various molecules function orthogonally and construct multiple signaling cascades even under a crowded environment. Cell behaviors have been regulated by cell signaling received from extracellular microenvironment via receptors and channel transporters. Metal ions are essential components of the cellular functions. It is estimated that around one-third of all proteins in living systems involve a metal ion for functionality. Especially, metalloenzymes, which are activated by specific metal ions, are indispensable to various biological processes by exhibiting versatile catalytic activities inside living cells. Recently in artificial cells, to induce dynamic cell behaviors such as signal transduction, cell division, or cell lysis, encapsulation of a metalloenzyme and regulation of its function inside the artificial cell have received increasing attention. However, regulation of cell behaviors by simultaneously loading of plural metalloenzymes have not been demonstrated yet even though metalloenzyme activation relies on a particular metal ion. In this respect, ionophores enable living cells to exchange selective ions with extracellular environment, resulted in regulation of diverse biological processes including metabolism, maintenance of osmotic pressure, regulation of cellular pH and signaling pathways. We here implemented to control behaviors of lipid-based single artificial cell encapsulating inactive plural metalloenzymes by influx of their cognate metal ions via ionophores on the cell membrane. As a result, our artificial cells exhibited dynamic cell behaviors such as pH increase leading to morphological transformation, activation of fluorophores, ROS generation, and cell lysis.
P3: A bottom-up approach to study front-rear cell polarization
Andreas Fink1, Charlotte Doll2, Sergio Lembo3, Anusha Gopalan3, Ana Yagüe-Relimpio1, Joachim Spatz1, Alba Diz-Muñoz3, Kerstin Göpfrich1, E. Ada Cavalcanti-Adam1
1: Max Planck Institute for Medical Research, Germany; 2: Heidelberg University, Germany; 3: EMBL, Germany
Spontaneous and induced front-rear polarization leading to cell movement is a key process involved in a variety of physiological and pathological events. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of polarization.
In living cells, in vitro systems based on surface micropatterning of adhesive proteins allow to impose reproducible changes in shape. Here we present a minimal model system for cell polarization using adherent Giant Unilamellar Vesicles (GUVs) on crossbow and line micropatterned surfaces. The GUV shape on micropatterns closeley resembles the shape of living cells adhering to similarly shaped patterns, suggesting that changes in the shape of polarised stationary cells are largeley governed by membrane tension and adhesion to the surface. To further study the effects of cytoskeletal organization on the GUV shape, actin filaments are polymerized together with bundling proteins inside the GUVs. Living cells adhering to micropatterns show a polarized cytoskeleton anchored to the membrane, whereas the distribution and orientation of actin filaments, not anchored to the membrane, in deformed GUVs appears to be random. In the future the GUV model system will be further developed to establish a mechanical linkage between the membrane and the cytoskeleton and to study forces involved in shape changes. As such, this bottom up approach will allow to identify the major components needed to create a polarized cytoskeleton, giving a better understanding of polarity in minimal systems.
P4: Engineering a synthetic cell wall using nanohybrid materials
Svetozar Gavrilovic
Max Planck Institute of Biochemistry, Germany
Although the initial cell precursors were compartmentalized only by early forms of membranes consisting of molecules such as lipids, it is unlikely that these membranes could have allowed life to evolve as it did. Protocells enclosed by such membranes would not have been robust enough and they wouldn’t be able to deviate from a spherical shape, characteristics that modern organisms possess and which are provided by the cell wall. Thus, the importance of cell walls and aspherical compartment geometries should be explored in the context of synthetic biology, as reconstituted protein systems could be studied in giant unilamellar vesicles (GUVs) that resemble living organisms. To choose building blocks for such synthetic cell walls one might turn to DNA origami due to its programmability and the possibility to crosslink into large-scale networks. By covering GUVs with self-assembled networks of DNA origami structures of different properties it would be possible to create synthetic coverings of different shapes and mechanical properties. To achieve different networks, the MinDE protein system could be used due to its ability to position membrane-bound molecules via diffusiophoresis. Membrane-bound DNA origami structures could be positioned on GUVs in different patterns depending on the concentration of MinD and MinE. With this added exoskeleton, potential shape deformation and its effect on different reconstituted protein systems, mechanical properties and the exchange between the GUVs and their surroundings will be explored. The development of such synthetic cell walls might also find various applications, as it could allow the positioning molecules of interest in drug delivery systems, as well as added robustness.
P5: In vitro regeneration of twenty aminoacyl-tRNA synthetases coupled with replication of artificial genomic DNAs
Katsumi Hagino, Norikazu Ichihashi
Department of Life Science, Graduate School of Arts and Science, The University of Tokyo, Japan
Autonomous self-reproduction is one of the fundamental functions of all living things. This function needs to be reconstituted in vitro for the complete construction of the artificial cell. The construction of autonomous reproduction system needs to combine the regeneration of enzymes required for gene expression with the replication of genetic information. However, such self-reproduction system has not been achieved due to low activity of the cell-free translation system and the complexity of the DNA replication system. In this study, we investigated whether artificial genomic DNAs encoding translation factors, 20 aminoacyl-tRNA synthetases (aaRS), replicate continuously through the expression of aaRSs from themselves in the reconstituted transcription/translation system of E. coli (PURE system) that lacks aaRSs. First, we found that each aaRS was expressed in a functional form in the PURE system and maintained the replication of the DNA encoding itself. By the similar method, we successfully continued the simultaneous replication of up to 5 aaRSs. Furthermore, by modifying sequence and employing dialysis mode reaction, we succeeded in the replication of all 20 aaRS DNAs with regenerating all 20 aaRSs. In this poster, we will report on the regeneration system developed in this study and discuss the next challenges for developing self-reproductive artificial cells.
P6: Pure protein bilayers and vesicles made from fungal hydrophobins: an alternative platform for synthetic cells
Hendrik Hähl1, Friederike Nolle1, Alessandra Griffo1, Kirstin Kochems1, Michael Lienemann2, Päivi Laaksonen3, Ralf Seemann1, Jean-Baptiste Fleury1, Karin Jacobs1
1: Department of Experimental Physics & Center for Biophysics, Saarland University, 66123 Saarbrücken, Germany; 2: VTT Technical Research Centre of Finland Ltd., Espoo 02150, Finland; 3: HAMK Tech, Häme University of Applied Sciences, Hämeenlinna 13101, Finland.
Compartmentalization of an aqueous solution is of utmost importance in biology. Typically, the matrix of the membranes forming the compartments is a phospholipid bilayer. Thereby, the amphiphilicity of the lipids is necessary for bilayer formation. Yet, for applications in, e.g., biomedicine or synthetic biology, phospholipids are limited in their variety in mechanical and biochemical properties and thus, alternative building blocks are needed. Proteins are promising candidates due to their biocompatibility and versatility via genetic engineering. A special family of strongly amphiphilic proteins, hydrophobins, appears to be particularly suited. In this study, we utilize fungal hydrophobins which self-assemble at water-interfaces into stable monolayer films. Contacting two interfacial films, stable bilayer membranes resembling lipid bilayers can be produced [1]. We study the assembly process of the monolayers and the mechanical properties of mono- and bilayers. At the interface, the proteins organize in clusters in which the proteins obtain a crystalline order [2]. In AFM membrane stretching experiments, we determine the elastic modulus of these monolayer films. We find a remarkably high value, very likely owing to the high cohesion in the 2D crystal structure. Yet, in the bilayer form, the modulus seems to be reduced, hinting at a protein reordering during bilayer formation.
References: [1] Hähl, H. et al., Adv. Mater 29, 1602888 (2017). [2] Hähl, H. et al., Langmuir 35, 9202 (2019).
P7: Opto-ParMRC: a minimal synthetic spindle with spatiotemporal control using optogenetic techniques
Yash Jawale, Reza Amini Hounejani, Koen van Gent, Eli van der Sluis, Marileen Dogterom
Delft University of Technology, The Netherlands
Growth and division are important features of a living systems. During division it is important that the DNA is segregated properly into the two daughter cells. Self-reproducing synthetic cells should be able faithfully segregate DNA for survival.
Here we present a design for a minimal spindle system based on the ParMRC system used for active segregation of R1 plasmid in Escherichia coli. In vivo ParM filaments form an antiparallel bipolar spindle which slides apart, where ParR connects the filament ends with centromeric parC plasmid DNA which promotes filament growth, thereby pushing sister plasmids to the cell poles. We aim to achieve temporal control over DNA segregation upon completion of DNA replication using optogenetic tools. We have split the filament- and DNA binding- domains of the ParR protein and linked them with the photo-switchable iLID-SspB protein pair.
This synthetic spindle will provide a minimal active DNA segregation system for a synthetic cell. The spatiotemporal control using photo-switchable proteins will help to impose external cell-cycle control. For this project, individual components have been designed and are currently being tested and quantified for their individual functions.
P8: Understanding codon optimality using redefined tRNA pools in vitro
Evan Kalb, Jose Alejo Amaya, Kate Adamala
University of Minnesota, United States of America
Aminoacylated tRNA abundance is a fundamental, if poorly understood, means of controlling the rate of protein translation. This gap in knowledge is likely due to challenge of precisely controlling tRNA abundances in vivo. To characterize the role of individual tRNA pools with minimal noise, we utilized a cell-free translation system to measure the rate of translation of a split luciferase with customized tRNA pools. Our data reveal that the translation machinery is highly sensitive to tRNA abundances and suggest noncognate tRNAs are inhibitory. We hope these findings may inform new considerations for codon optimality in in vitro systems and may realize greater efficiency in cell-free translation.
P9: Bottom-up reconstitution of the bacterial division system in synthetic cells via cell-free expression
Shunshi Kohyama, Adrián Merino-Salomón, Petra Schwille
Max Planck Institutite of Biochemistry, Germany
Reconstitution of minimal cell division processes in lipid vesicles is a crucial step towards bottom-up construction of a synthetic cell. In this regard, one of the most studied systems is the division machinery of Escherichia coli, in which Min proteins exhibit spatial-temporal gradient, so-called Min waves, targeting the FtsZ protein into a ring-like structure at mid-cell. Especially, reconstitution of these division machinery via cell-free protein synthesis systems allows imitating life-like systems in vitro, although this approach is still challenging due to the complexity of the expression of multiple functional proteins at the same time. Indeed, several of these division machineries have been reconstituted inside lipid compartments by using not only purified proteins but also cell-free expression systems. However, the full sequence of self-organizing processes of division proteins has not been reconstituted yet.
Here, we demonstrated the reconstitution of the self-organization process of E. coli division machinery within lipid vesicles via cell-free expression of five essential division proteins, namely, MinC, MinD, MinE, FtsA, and FtsZ. By employing the long time-lapse imaging technique under the thermo-control unit, we were able to observe the sequence of self-organization events, such as formation of FtsZ-mesh, emergence of Min waves, and placement of a FtsZ-ring structure. Remarkably, we further observed a radial constriction of lipid vesicles, suggesting the significant force generation by a FtsZ-ring. Our results showed an exceptional example of such a complex spatial-temporal biological system reconstituted in vitro, providing a key step towards emerging of cell division in a synthetic cell.
P10: Bottom-up assembly of functional fully synthetic extracellular vesicles for atopic dermatitis treatment
Meline Macher1, Rahul Rimal1, Smriti Singh1, Rin Ho Kim2, Assa Yeroslaviz2, Eva Bertosin3, Nils Chapin1, Karen Bieback4, Ilja Platzman1, Joachim P. Spatz1
1: Max Planck Institute for Medical Research, Germany; 2: Max Planck Institute of Biochemistry, Germany; 3: TU Munich, Germany; 4: University Medical Centre Mannheim, Germany
Extracellular vesicles (EVs) have been recently accepted as pivotal contributors for intercellular communication, carrying a plethora of bioactive molecules such as soluble and membrane proteins, nucleic acids and metabolites. Many studies have shown beneficial therapeutic effects of different EVs against cardiovascular, immune and skin diseases. However, despite their promising clinical outcomes, there are currently no EV-therapeutics approved by the regulatory authorities. This is mainly attributed to the fact that therapeutic EVs are isolated from cell culture conditioned medium, which is intrinsically subjected to natural batch-to-batch variations and contaminations, leading to limited scalability and high time effort and cost of isolation. Moreover, natural EVs cannot be precisely engineered on the molecular level, hindering mechanistic insights into EV mechanisms of action. In our research, we apply principles and technologies from bottom-up synthetic biology and biomaterial engineering for the controlled bottom-up assembly of fully-synthetic EVs (synEVs) that comprise similar physiological functionalities to natural human mesenchymal stem cell derived (hMSC) EVs. We assess in vitro the therapeutic capabilities of assembled synEVs and natural hMSC EVs in atopic dermatitis skin models, a chronic inflammatory skin disease with rising prevalence and limited treatment options. The developed approach opens the door for EV-based therapies that do not rely on the isolation of EVs from natural sources but rather on bio-inspired fully-synthetic EVs that will allow for testing structure-function relationships of individual EV components in a quantitative manner.
P11: Developing anti-quorum communication coatings by embedding SynCells in porous silica thin films
Aidira D.Y. Macias Gonzalez1,2, Jacqueline De Lora2, Ashley N. Tafoya1, Matthew L. Lakin1, Joachim P. Spatz2, Gabriel P. Lopez1
1: The University of New Mexico, United States of America; 2: Max Planck Institute for Medical Research, Germany
Bacterial colonization often employs quorum sensing communication between microbial cells. Such biochemical communication may offer a means to sense the presence of pathogenic bacteria on the surface of an implant, and, in principle, to respond to such threats as well. Giant unilamellar vesicle (GUV)-based SynCells entrapped in porous, thin silica films offer this possibility. We describe the fabrication of hybrid materials using biofriendly sol-gel chemistry for biomedical applications. Our long-term goal is to develop porous silica coatings that incorporate synthetic cells (SynCells) that can sense and respond to exogenous biochemical cues. To produce porous materials, we use tetramethyl orthosilicate as a precursor, with a low molecular weight polyethylene glycol to conserve the integrity of rudimentary models for SynCells entrapped within the silica thin film. The sol-gel chemistry employed differs from conventional methods as it does not use methanol or other alcohols as a cosolvent and potentially harmful catalysts and byproducts. Vaporization is used to eliminate the methanol formed during silica condensation in the sol-gel process. We then entrap SynCells, comprising GUVs, to preserve their stability for long periods of time and show that this further depends on storage temperature of either 20 or 4°C. SynCells entrapped in silica matrices exhibit long-term stability especially when stored at lower temperature. We present easy to fabricate hybrid, thin, porous silica matrices that both extend the structural integrity of embedded GUV-based SynCells and offer the ability to interact with small molecules that can freely diffuse through the porous silica matrix.
P12: In vitro assembly of minimal cell division proteins inside lipid vesicles
Adrian Merino Salomon, Shunshi Kohyama, Petra Schwille
Max Planck Institute of Biochemistry, Germany
One of the major goals in the bottom-up synthetic biology is the construction of a minimal machinery for autonomous self-division. In order to reassemble such a division system in vitro, a strong focus has been on the E. coli division system, which is driven by the assembly of a presumably contractile ring-like structure based on the tubulin homologue FtsZ attached to the membrane through the interaction with its membrane adaptor FtsA or ZipA. Interestingly, the formation and positioning of this ring-like structure at midcell is essentially mediated by self-organization of the MinCDE system following a pole-to-pole oscillation, avoiding the assembly of the FtsZ-ring at cell poles, allowing cytokinesis and eventually cell division. Here, we demonstrated the full assembly of a minimal cell division machinery inside cell-like containers using giant unilamellar vesicles (GUVs). Employing the purified components, we co-reconstituted a minimal division machinery composed by a functional MinCDE system in combination with a FtsZ chimeric protein, assembling and positioning a ring-like structure at the equatorial plane of a cell-like model. We highlight the importance of critical experimental factors, such as physiological buffer, macromolecular crowding and confinement, which play an important role in vivo. Our results provide an exceptional example of the emergence of bacterial cell division in a minimal system, being one of the first steps towards the achievement of an in vitro self-division.
P13: Molecular cues trigger Boolean response in programmable protocells
Ananya Mishra, Avinash Patil, Stephen Mann
University of Bristol, United Kingdom
Living cells are smart autonomic machines that can recognise, sort and process complex molecular cues and respond to their environmental changes to exhibit higher order functionalities such as metabolism, growth, division, motility etc. Construction of synthetic cells/protocells integrated with biomimetic functions such as membrane gating, molecular crowding and spatially controlled enzyme cascades driven chemical signal processing are providing opportunities to engineer non-living materials with life-like properties.
Herein we have designed complex coacervate droplets (protocells) derived from non-covalent interactions between oppositely charged polyelectrolytes and nucleotide that mimic a molecularly crowded interiors within cells. Significantly, these membrane free protocells can sequester high concentrations of range of biotic and abiotic functional molecules and therefore can be employed as programmable protocells to achieve bioinspired functions. We show that the highly ordered microarrays of the coacervates installed with wide range of multi-enzyme cascades can receive, sort and process input chemical signals to execute range of Boolean logic functions. Significantly, the protocell-based Boolean logic operations were further advanced by establishing communication between the spatially separated populations of coacervates. Such collective information processing gives rise to opportunities to create feedback loops for programmable output generation. We envisage that protocell-based logic gates will provide opportunities to construct complex biocomputing devices for rapid diagnostics and clinical applications.
P14: VASP phase separates on lipid bilayers to induce polymerization driven actin bundle formation
Timon Nast-Kolb, Andreas Bausch
Technische Universität München, Germany
Actin bundles constitute important cytoskeleton structures, enabling a scaffold for force transmission inside cells. Actin bundles are formed by proteins with multiple F-actin binding domains crosslinking actin filaments to each other. VASP is mostly reported as an actin elongator, but has been shown to be a bundling protein as well. It is localized in bundled actin structures in filopodia, adhesion sites and stress fibers. From in vitro experiments, it remains unclear when and how VASP can act as an actin bundler or elongator. We show that VASP bound to supported lipid bilayers facilitates the formation of actin bundles during polymerization. Elongating filaments on the bilayer are captured by VASP at bundles and continuously elongated directly aligned to the bundle. This results in a pronounced growth of the bundle thickness. This alignment by polymerization requires the fluidity of the lipid bilayers. The mobility within the bilayer enables VASP to capture and track growing barbed ends or freely move on the sides of filaments. VASP itself can phase separate on the bilayer at high concentrations. The VASP enriched phase then accumulates underneath the actin bundles during polymerization. This binding to the actin bundles of VASP results in turn in a reorganization of the underlying lipid bilayer. Our findings demonstrate that the nature of VASP localization is decisive for its function. The upconcentration based on its affinity to actin during polymerization enables VASP to fulfil the function of an elongator and bundler simultaneously.
P15: Extracellular matrix-based protein microcapsules for potential use as advanced drug delivery system
Roxana Pashapour1, Sadaf Pashapour1, Senne Seneca1, Claus Kordes2, Petra May2, Hans Bock2, Ilia Platzman1, Joachim Spatz1
1: Max-Planck-Institute for Medical Research, Institute Molecular Systems Engineering (IMSE, Heidelberg), Germany; 2: Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Düsseldorf, Germany.
Structural remodelling of the extracellular matrix (ECM) by cancer cells has an impact on tumor progression and metastasis. Cancer cells make use of ECM components, such as laminin-111 (LN) proteins, to establish a protective microenvironment towards the immune system and several chemotherapeutics. In this study, we hypothesize that the production of LN-based microcapsules improves the delivery of chemotherapeutics to cancer cells by enhancing its penetration and uptake into the cancerous ECM. We establish doxorubicin-loaded LN microcapsules using water-in-oil emulsion droplets consisting of negatively charged block-copolymer surfactants. Charge-mediated formation of LN scaffolds are then generated at the water-oil interface, templating the capsules using the inner surface of the droplet. After removal of the oil phase, a breast cancer cell line (MCF-7) is incubated with the microcapsules and their potency against MCF-7 cells is evaluated. Preliminary results show that MCF-7 cells interact with doxorubicin-loaded microcapsules, which triggers a localized and slow cytotoxicity towards MCF-7 cells in direct contact with the capsules. In conclusion, these results suggest that by encapsulating doxorubicin in ECM-based protein microcapsules we improve specific interactions between cancer cells and the drug-loaded protein microcapsules. This new approach can reduce the drug concentration needed to exert antitumorigenic effects and may prevent unspecific and uncontrolled killing of normal cells through free doxorubicin. Future applications, what we investigate here for drug delivery, could include using the capsules to provide a realistic extracellular environment for a synthetic cell.
P16: DNA secondary structure formation trigger phase separation of membrane less organelles
Federica Raguseo, Marco Di Antonio, Lorenzo Di Michele, Yuval Elani
Imperial College London, United Kingdom
It is widely recognised that cells regulate the formation of membrane-less organelles via liquid-liquid phase separation [1]. Less well defined, however, is the exact characterisation of such physical phenomenon. Part of the answer resides in the presence of RNA and RNA-binding proteins with a high concentration of low complexity domains whose interactions have been hypothesized to lead to phase separation [2]. Although historically this field has mainly focused on protein-led separation, especially in the case of non-functional pathological aggregates involved in neurodegenerative diseases, recent studies showed that non-coding RNAs could equally contribute to formation of phase-separated aggregates [3].
In this work I investigate the chemo-physical properties of the repeat sequence (GGGGCC)n, taken from the C9orf72 gene and correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [4]. Both these diseases are characterised by the presence of pathological aggregates similar in composition to membrane-less organelles. Interestingly, (GGGGCC)n has been found in phase-separated condensates and has been shown to form G-quadruplexes (G4s), an alternative DNA secondary structure generated from non-canonical base pairing between guanines.
I discovered a correlation between G4s formation and nucleic acid aggregation in absence of any protein, offering a new insight on understanding phase separation in cells and on the progression of these neurodegenerative diseases by targeting the disassembly of the G4 itself.
References: [1]. Gomes E et al, Biol Chem. 2019; [2]. Banani SF et al, Nat Rev Mol Cell Biol. 2017; [3]. Garcia-Jove Navaro M et al, Nature Comm. 2019; [4]. Iacoangeli A et al, Acta Neuropathologica Comm. 2019.
P17: In vitro reconstitution of glycolysis-driven protein synthesis
Gaku Sato, Saki Kinoshita, Takahiro Yamada, Akira Funahashi, Nobuhide Doi, Kei Fujiwara
Keio University, Japan
One of the important goals of bottom-up synthetic biology is to reconstitute living cells from defined factors. So far, essential biochemical systems, such as glycolysis, transcription-translation (TX-TL), genome replication, and phospholipid synthesis, have been reconstituted in vitro. Although integration of these systems will lead to reconstitution of living cells, there are few research focusing on conjugation of individually reconstituted systems. Here, we reconstituted the conjugated system of glycolysis and TX-TL, and elucidated the condition under which their coupling occurs. Compared with the case of cell extract, initial condition for coupling was narrower in our purified system, suggesting that there is unknown mechanism that promotes coupling in extract. Based on this hypothesis, we explored the mechanism and revealed that irrelevant ATP-consuming system widened the coupling condition through adjusting the unbalance between ATP regeneration of glycolysis and ATP consumption of TX-TL. These results indicate that proper distribution of common elements is critical for coupling of biochemical systems. Our findings will contribute to understand how biochemical systems avoid crosstalk inside living cells. Moreover, because glycolysis and transcription-translation are the core metabolism that synthesize important biomolecules including energy, amino acids, and protein, our coupling system will be the basis for artificial cells.
P18: Towards functional SecYEG reconstitution in giant unilamellar vesicles
Ludo Schoenmakers1, Max den Uijl2
1: Radboud University, The Netherlands; 2: University of Groningen, The Netherlands
One of the key challenges in building a synthetic cell is membrane protein insertion. Without the machinery for transmembrane protein insertion, the various membrane proteins that are critical for metabolite uptake, cell volume regulation, lipid biogenesis, and a host of other processes, cannot be inserted. The system responsible for the bulk of membrane protein insertion at the bacterial inner membrane is the Sec translocon. Here, we show our work on reconstituting a minimal version of the Sec translocon into giant unilamellar vesicles. Our ultimate aim is to show that this minimal Sec system is able to translocate proteins across the membrane, before moving on to the co-translation insertion of an active membrane protein.
P19: Liposome preparation by 3D-printed microcapillary-based apparatus
Orion M. Venero, Wakana Sato, Joseph M. Heili, Christopher Deich, Katarzyna P. Adamala
University of Minnesota, United States of America
Liposomal encapsulation serves as the basis for the engineering of biomimetic and novel synthetic cells. Liposomes are normally formed using such methods as thin film rehydration (TFH), density mediated reverse emulsion encapsulation (REE), or one of many microfluidics-based approaches. While several microfluidics-based methods exist, capable of efficiently forming unilamellar liposomes, with uniform size and acceptable encapsulation rates, the main limitation associated with microfluidics is that trace amounts of carrier solvent inevitably remain present in the resultant membrane. A popular method bypasses the problem of residual solvent presence by first, evaporating all carrier solvent: thereby creating a thin lipid film used to prepare liposomes through subsequent methods. However, most protocols which utilize this methodology of thin film preparation rely upon ‘uncontrolled’ rehydration and thus fail to produce uniform results.
DSCF, which stands for Droplet‐Shooting Centrifugal Formation, is a derivative liposome formation method that utilizes solvent-free lipid thin films to prepare highly uniform lipid bilayer membranes using a 3D printable microfluidics system. In this way, DSCF methods avoid the solvent-related issues associated with microfuidics while producing liposomes with a high level of repeatability, similarly to microfluidics. The DSCF device described herein may be 3D printed using an SLA printer or an online 3D printing service. Additionally, the Adamala Lab developed an injection moldable version for the production of micro capillary collets in Polyetherimide (PEI).
P20: First steps towards mimicking cell division in dendrimersome synthetic cells
Anna M. Wagner1, Hiromune Eto2, Anton Joseph1, Shunshi Kohyama2, Tamas Haraszti1, Ricardo Zamora3, Mariia Vorobii1, Marina Giannotti3, Petra Schwille2, Cesar Rodriguez-Emmenegger1,3,4
1: DWI Leibniz Institute, Germany; 2: Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Germany; 3: Institute for Bioengineering of Catalonia (IBEC), Spain; 4: Institució Catalana de Reserca I Estudis Avançats (ICREA), Spain.
The integration of active cell machinery with synthetic building blocks is the bridge towards developing synthetic cells with functions beyond life. In nature, the E. coli divisome is one of the simplest yet extremely elegant ways to divide cells. FtsZ proteins assemble into the Z-ring exerting a force to sever the membrane. The Z-ring must be positioned at the equator, a task that is controlled by the Min system. Currently, the Min system has only been reconstituted in liposomes presumably due to the large differences in physical properties of synthetic cell membranes. This seriously limits the applications in which these synthetic cells can be used. Here we show new biomimetic dendrimersomes assembled from zwitterionic Janus dendrimers supporting the reconstitution of an active bacterial divisome. This was possible by programming the strength and dynamics of membrane-divisome interactions and reproduce its dynamic behaviour. This constitutes a breakthrough in the assembly of synthetic cells with biological elements, as tuning of membrane-divisome interactions is the key to engineer emergent biological behaviour from the bottom-up.
P21: Understanding the pancake-like morphology of the Golgi Apparatus compartments
Gianna Wolfisberg1, Henrik Spanke1, Toshikaze Chiba2, Naohito Urakami3, Alexandre Torzynski1, Masayuki Imai2, Eric Dufresne1, Aleksander Rebane1
1: Department of Materials, ETH Zürich, Switzerland; 2: Department of Physics, Tohoku University, Japan; 3: Department of Physics and Informatics, Yamaguchi University, Japan
The Golgi Apparatus is a highly conserved membrane organelle involved in a complex cascade of protein post-translational modifications, with function being closely tied to morphology. Based on bending energy minimisation, one would expect membrane compartments of low volume-to-surface ratios, as found in the Golgi compartments (cisterna), to form a cup-like stomatocyte. In nature, however, the Golgi compartments have a flat pancake-like morphology. We investigate additional factors required for this flat pancake-like morphology. Therefore, we adhere giant unilamellar vesicles (GUVs) of varying volume-to-surface ratios to flat surfaces with varying adhesion energies. We find that by combining bending energy with adhesion, pancake-like shapes at lower volume-to-surface ratios can be achieved. However, it does not result in the ultra-flat morphology observed in the Golgi, implying that factors additional to bending and adhesion energy, such as the presence of rim stabilising proteins or lipids need to be considered.
P22: Interactions among distinct surface-adhered protocell species
Lin Xue, Irep Gözen
University of Oslo, Norway
We report on the physico-chemical interactions between protocells spontaneously formed from distinct molecular lipid films on solid surfaces. By forming two types of solid-supported membranes from different phospholipids and fluorophores, we were able to monitor the emergence of spherical protocell populations from each lipid film, and the subsequent interactions among them such as fusion, mixing and migration. Supported lipid membranes can mix upon contact, and in hypotonic solutions spontaneously generate compartments in varying ratios of the original lipids, leading to compositionally new species of protocells. When protocells are exposed to an elevated calcium concentration, multi-layered lipid structures form that can migrate over several tens of micrometers with velocities reaching 0.3 μm/s. By introducing RNA oligomers to the proximity of protocells in presence of calcium, we observed the simultaneous adsorption and encapsulation of RNA. The RNA adsorbed on the protocellular membranes can relocate during the topological transformations of the lipid structures. Our findings show how rudimentary interactions among different primitive cell populations could occur on solid surfaces which can lead to generation of dynamic protocell species that are compositionally diverse without dividing.
P23: Bottom-up assembly of synthetic SARS-CoV-2 minimal virions for the investigation of viral infectivity
Ana Yagüe Relimpio1, Oskar Staufer2, Andreas Fink1, Alessia Ruggieri3, Imre Berger4, Ilia Platzman1, Joachim Spatz1
1: Max Planck Institute for Medical Research, Germany; 2: University of Oxford, United Kingdom; 3: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Germany; 4: Max Planck-Bristol Center for Minimal Biology, University of Bristol, United Kingdom
SARS-CoV-2 is the betacoronavirus responsible for the still ongoing COVID-19 pandemic, which still remains a major global health concern. Due to the high mutagenicity of the virus, well-controlled studies of its infectivity are challenging. Furthermore, performing research with natural viruses is often dangerous and complex, and they are restricted to high biosafety facilities. We developed a technology for the bottom-up assembly of modular minimal SARS-CoV-2-like virions with molecularly defined composition. The bottom-up synthesis of synthetic virions consisted of the creation of small unilamellar vesicles (SUVs) with a lipid composition that resembles that of the SARS-CoV-2 virus. The vesicles were further functionalized with spike glycoprotein. Under low biosafety conditions we performed a systematic screening of spike-functionalized SUV binding to target cells under well-defined free fatty acid conditions. We found that unsaturated fatty acid binding to the spike reduced spike-mediated SARS-CoV-2 infectivity. Importantly, our technology could be modulated to study the infectivity of different SARS-CoV-2 variants of concern, as well as to assess the direct impact of FDA-approved drugs on spike protein cell-binding activities. Additionally, we aim to decipher the effect of the lipid composition – both on the cell and on the virus membrane – on viral infectivity. Specifically in the case of SARS-CoV-2, the importance of cholesterol and sphingomyelin have been highlighted as a key element for the infectivity of the virus. The developed technology allowed us to circumvent the current limitations associated with studies of natural SARS-CoV-2 viruses for potential therapeutical applications.
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